Journal: Transfusion Medicine and Hemotherapy

Article Title: Inter-Laboratory Comparison of Extracellular Vesicle Isolation Based on Ultracentrifugation

doi: 10.1159/000508712

Figure Lengend Snippet: FACS-based characterization of isolated EVs (second round). A FACS histograms depicting the relative fluorescence/marker intensity of EV preparation 2.1 (black line) against unstained EV particle control (grey line). B Corresponding marker expression in HCT116 cells (extracellular staining for CD9, CD63, and CD81 and intracellular staining for Alix, TSG101, and calnexin). C, E Mean fluorescence intensity (MFI) raw values of CD63 (C) and CD81 (E) marker expression from laboratories 2.1–2.4. D, F MFI values per particle concentration of CD63 (D) and CD81 (F; left y axis) against the respective particle concentration per ml CCM (right y axis).

Article Snippet: HCT116-derived EVs were captured on anti-human CD9 beads for flow detection (Exosome-Human CD9 beads; Thermo Fisher Scientific).

Techniques: Isolation, Fluorescence, Marker, Control, Expressing, Staining, Concentration Assay

Journal: Physiological reports

Article Title: Circulating extracellular vesicle characteristics differ between men and women following 12 weeks of concurrent exercise training.

doi: 10.14814/phy2.16016

Figure Lengend Snippet: FIGURE 3 EV surface marker expression is altered by AHRET in men and women. The proportion of EVs expressing SGCA (a), CD9 (b), VAMP3 (c), and THSD (d) were measured via Imaging Flow Cytometry and are shown as percentage of total gated EVs. Representative images of EV particles with bright-field (BF) image of each particle and subsequent fluorescent channel images showing presence or absence of muscle-derived EVs (SGCA+), microvesicles (VAMP3+), exosomes (CD9+) and apoptotic bodies (THSD+) vesicles are shown (e). N = 9. Statistical testing was done via three-way ANOVA.

Article Snippet: Samples were then stained with the following antibodies and dilutions: anti- human CD9 Alexa Fluor 700 (1:300 dilution; Novus Biologicals, CO), anti- human VAMP3 Alexa Fluor 405 (1:300; Novus Biologicals), anti- human thrombospondin (THSD- 1) Alexa Fluor 594 (1:100; Novus Biologicals), alpha sarcoglycan (SGCA) FITC (1:400; Biorbyt, St Louis, MO).

Techniques: Marker, Expressing, Imaging, Flow Cytometry, Derivative Assay

Journal: Non-Coding RNA

Article Title: Stabilization of Urinary MicroRNAs by Association with Exosomes and Argonaute 2 Protein

doi: 10.3390/ncrna1020151

Figure Lengend Snippet: Enrichment of urinary miR-16, miR-192 and classical exosomal markers in the same sucrose gradient fractions. Following overnight sucrose gradient ultracentrifugation of the sedimented urinary pellet at 210,000 g at 4 °C, enrichment was observed in sucrose gradient fractions 3–5 of ( a ) multivesicular body markers TSG101 and Alix detected by Western analysis ( b ) exosome-expressed tetraspanins CD9 and CD81 detected by flow cytometry and ( c ) RT-qPCR-detected miR-16 and miR-192.

Article Snippet: Primary monoclonal antibodies (2–10 μg/mL) against CD9 (R&D Systems, Abingdon, Oxfordshire, UK) and CD81 (AbD Serotec, Kidlington, Oxfordshire, UK) were used for 1 h at room temperature.

Techniques: Western Blot, Flow Cytometry, Quantitative RT-PCR

Journal: Biology

Article Title: Characterization of Rabbit Mesenchymal Stem/Stromal Cells after Cryopreservation.

doi: 10.3390/biology12101312

Figure Lengend Snippet: Figure 5. Representative histograms of MSC surface marker expression of CD9 and CD44, and hematopoietic marker expression of CD34 and CD45. The red histogram represents isotype control, and the green histogram represents respective antibodies. MSCs, mesenchymal stem/stromal cells; ADP, adipose tissue; BM, bone marrow; BF, before cryopreservation; AF, after cryopreservation.

Article Snippet: CD9 (catalog no. NBP1-28364, Novus Biologicals, Centennial, CO, USA) and CD44 (catalog no. bs-0521R-FITC, Bioss, Woburn, MA, USA) were used as MSC surface marker antibodies, and CD34 (catalog no. bs-0646R-FITC, Bioss, Woburn, MA, USA) and Biology 2023, 12, 1312 5 of 23 CD45 (catalog no. MHCD450, Thermo Fisher Scientific Inc., Life Technologies Corporation, Frederick, MD, USA) were used as hematopoietic cell marker antibodies [40–44].

Techniques: Marker, Expressing, Control

Journal: Alzheimer's Research & Therapy

Article Title: Immuno-digital invasive cleavage assay for analyzing Alzheimer’s amyloid ß-bound extracellular vesicles

doi: 10.1186/s13195-022-01073-w

Figure Lengend Snippet: Immuno-digital ICA (idICA) for counting Aß-bound EVs. A , B Workflow of immune-digital ICA analysis of Aß-bound EVs. Ganglioside GM1-containing EVs are captured by cholera toxin B subunit (CTB)-coated magnetic beads (MB) and then reacted with the DNA oligo-conjugated detection antibody against an exosome marker protein CD9 or Aß ( A ). The resultant EV–bead–antibody complex in A and substrates for ICA are loaded into the digital device, enclosed into individual microwells by fluorinated oil, and analyzed by fluorescent imaging after the ICA reaction at 66 °C for 15 min ( B ). C Schematic illustration of ICA. (1) Invasive oligonucleotides and probe oligonucleotides hybridize to target DNA and generate 5′-flap structures in the probe oligonucleotides, which are cleaved by FEN-1. The target DNA is conjugated to detection antibodies. (2) The cleaved 5′-flaps bind to fluorescent probes and form 5′-flap structures between a quencher molecule [Q] and a fluorophore [F]. Cleavage of 5′-flaps by FEN-1 emits fluorescence signals. Unannealed 5′-flaps are shown in blue and yellow. Arrows indicate 5′-flap cleavage by FEN-1. D The digital device used in this study. There are 100 blocks of well arrays; 10,000 wells in each block correspond to the 10 6 microwells on a single fabricated device. Phase-contrast image and fluorescent image of a block of well array are shown. Scale bars, 1 mm

Article Snippet: Monoclonal antibodies against CD9 (MAB5218, R&D Systems, Minneapolis, MN), Aß (BAN50, FUJIFILM, Tokyo, Japan), and ßIII tubulin (#2146, Cell Signaling, Danvers, MA) were used.

Techniques: Magnetic Beads, Marker, Imaging, Fluorescence, Blocking Assay

Journal: Alzheimer's Research & Therapy

Article Title: Immuno-digital invasive cleavage assay for analyzing Alzheimer’s amyloid ß-bound extracellular vesicles

doi: 10.1186/s13195-022-01073-w

Figure Lengend Snippet: Quantification of GM1-containing EVs Using the idICA. A Western blot analysis of CD9, ganglioside GM1, and ßIII tubulin in N2a cell lysates (1 × 10 5 cells/lane) and EVs (1 × 10 7 cells/lane). B Representative fluorescent images of various concentrations of N2a-derived EVs in the idICA, which is constructed from CTB capture and anti-CD9 detection. Each image shows a block of well array corresponding to 10,000 microwells. Scale bar, 200 μm. C , D The ratio of fluorescent beads to trapped beads in a block of well array is plotted as the concentration of CD9 captured on CTB-coated beads (CTB-CD9) in N2a-derived EVs. Plots on the semi-logarithmic ( C ) and linear ( D ) scales are shown. Data represent mean ± SD ( n = 3 each)

Article Snippet: Monoclonal antibodies against CD9 (MAB5218, R&D Systems, Minneapolis, MN), Aß (BAN50, FUJIFILM, Tokyo, Japan), and ßIII tubulin (#2146, Cell Signaling, Danvers, MA) were used.

Techniques: Western Blot, Derivative Assay, Construct, Blocking Assay, Concentration Assay

Journal: Alzheimer's Research & Therapy

Article Title: Immuno-digital invasive cleavage assay for analyzing Alzheimer’s amyloid ß-bound extracellular vesicles

doi: 10.1186/s13195-022-01073-w

Figure Lengend Snippet: Quantification of Aß-bound and GM1-containing EVs using the idICA. A Western blot analysis of Aß, ganglioside GM1, and ßIII tubulin in APP-N2a cell lysates (1 × 10 5 cells/lane) and EVs (1 × 10 7 cells/lane). B Representative fluorescent images of various concentrations of APP-N2a-derived EVs in the idICA, which is constructed from CTB capture and anti-Aß detection. Each image displays a block of well array corresponding to 10,000 microwells. Scale bar, 200 μm. C , D The ratio of fluorescent beads to trapped beads in a block of well array is plotted as the concentration of Aß captured on CTB-coated beads (CTB-BAN50) in APP-N2a-derived EVs. Plots on the semi-logarithmic ( C ) and linear ( D ) scales are shown. Data represent mean ± SD ( n = 3 each). E Representative images of APP-N2a-derived EVs (2500 ng protein) in the double color idICA using anti-CD9 antibody and BAN50. Each image displays a block of well array corresponding to 10,000 microwells. Scale bar, 200 μm. F The ratio of BAN50 or CD9 fluorescent beads to trapped beads. G The overlap rate between BAN50 and CD9 fluorescent beads. Data represent mean ± SD ( n = 5 each)

Article Snippet: Monoclonal antibodies against CD9 (MAB5218, R&D Systems, Minneapolis, MN), Aß (BAN50, FUJIFILM, Tokyo, Japan), and ßIII tubulin (#2146, Cell Signaling, Danvers, MA) were used.

Techniques: Western Blot, Derivative Assay, Construct, Blocking Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Opportunities and Pitfalls of Fluorescent Labeling Methodologies for Extracellular Vesicle Profiling on High-Resolution Single-Particle Platforms

doi: 10.3390/ijms221910510

Figure Lengend Snippet: Characterization of purified sEV subpopulations based on their surface markers. ( A ) Antibody staining efficiency was evaluated by nFCM using anti-CD9 antibodies conjugated with different fluorophores. ( B ) HT29 and HEK293 sEVs were stained with anti-tetraspanin (CD9, CD63, and CD81) antibodies, conjugated with either PE or AF488, and analyzed on nFCM ( n ≥ 3; mean ± SEM). ( C ) Cross-platform and inter-batch variability was assessed by single-staining HT29 sEV (batches #A and #B) with anti-tetraspanin AF488 antibodies ( n = 3; mean ± SEM). Differences in tetraspanin expression between nFCM and F-NTA, and batches #A and #B, were assessed using two-way ANOVA with Tukey’s test for multiple comparisons (alpha = 0.05, p = 0.1234 (ns), 0.0002 (***), <0.0001 (****)). Detailed results of the statistical analysis are provided as . ( D ) To evaluate single, as well as co-expressing, events, HT29 sEVs were stained with a mix of 2 and 3 different anti-tetraspanin AF488 antibodies and analyzed on F-NTA ( n = 3; mean ± SEM), or on ( E ) nFCM using PE-conjugated antibodies ( n = 3; mean ± SEM). HT29 sEV subpopulations expressing either 1, 2, or 3 markers are represented in the Venn diagram on the left. The Venn diagram on the right refers to the same HT29 sEVs, however it depicts subpopulations co-expressing both 2 or 3 markers simultaneously. Additional data from procedural controls, as well as the F-NTA PSD histograms, are provided in .

Article Snippet: The following fluorescently-labeled primary antibodies were used: Phycoerythrin (PE)-conjugated mouse anti-human CD9, CD63 and CD81 (dilution 1:10 for all; Exbio, Vestec, Czech Republic), Alexa Fluor ® 488 (AF488)-conjugated mouse anti-human CD9, CD63 and CD81 (1:500, 1:25 and 1:500, respectively; R&D Systems, Minneapolis, MN, USA), allophycocyanin (APC)-conjugated mouse anti-human CD9 (1:10; Exbio), Alexa Fluor ® 647 (AF647)-conjugated mouse anti-human CD9 (1:10; Exbio).

Techniques: Purification, Staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Opportunities and Pitfalls of Fluorescent Labeling Methodologies for Extracellular Vesicle Profiling on High-Resolution Single-Particle Platforms

doi: 10.3390/ijms221910510

Figure Lengend Snippet: Suitability of SEC and UF as methods for clearing dyes in excess after sEV fluorescent labeling and their effect on subpopulation ratios. ( A ) Comparison of labeling % for stained HT29 sEVs before and after the removal of excess dye. 1 × 10 9 or 5.5 × 10 9 sEVs were incubated with antibodies (aCD9 1:12.5; aCD63 1:12.5; aCD81 1:25) or CFSE (50 µM), respectively, and measured by F-NTA. To remove the excess dye, UF washing strategy was applied, followed by F-NTA detection. ( B ) To compare different strategies for the removal of fluorescent antibodies in excess, labeling %, ( C ) PSD histograms, and ( D ) median and mean particle diameter were assessed for CD9+, CD63+ and CD81+ HEK293 sEVs, on nFCM. 2 × 10 9 sEVs, at a concentration of 10 8 particles/µL, were incubated with PE-labelled antibodies (aCD9 1:500; aCD63 1:25; aCD81 1:500) and unbound antibodies were removed by SEC or UF. Sample dilution (500–1000-fold) served as staining reference. Data is presented as mean ± SEM of at least three independent experiments. F-NTA PSD histograms of samples analyzed before and after washing are provided in .

Article Snippet: The following fluorescently-labeled primary antibodies were used: Phycoerythrin (PE)-conjugated mouse anti-human CD9, CD63 and CD81 (dilution 1:10 for all; Exbio, Vestec, Czech Republic), Alexa Fluor ® 488 (AF488)-conjugated mouse anti-human CD9, CD63 and CD81 (1:500, 1:25 and 1:500, respectively; R&D Systems, Minneapolis, MN, USA), allophycocyanin (APC)-conjugated mouse anti-human CD9 (1:10; Exbio), Alexa Fluor ® 647 (AF647)-conjugated mouse anti-human CD9 (1:10; Exbio).

Techniques: Labeling, Comparison, Staining, Incubation, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Opportunities and Pitfalls of Fluorescent Labeling Methodologies for Extracellular Vesicle Profiling on High-Resolution Single-Particle Platforms

doi: 10.3390/ijms221910510

Figure Lengend Snippet: Multiplex fluorescence analysis for enhanced sEV identification and characterization. ( A ) Purified HT29 sEVs were labeled with anti-CD9-AF488 and anti-CD81-PE either individually (single staining) or in combination (double staining). ( B ) HT29 sEVs were stained with CTR and anti-CD81-AF488 either individually or in combination. Results are shown as % of labeled particles detected on nFCM, in two fluorescence channels ( n = 3; mean ± SEM).

Article Snippet: The following fluorescently-labeled primary antibodies were used: Phycoerythrin (PE)-conjugated mouse anti-human CD9, CD63 and CD81 (dilution 1:10 for all; Exbio, Vestec, Czech Republic), Alexa Fluor ® 488 (AF488)-conjugated mouse anti-human CD9, CD63 and CD81 (1:500, 1:25 and 1:500, respectively; R&D Systems, Minneapolis, MN, USA), allophycocyanin (APC)-conjugated mouse anti-human CD9 (1:10; Exbio), Alexa Fluor ® 647 (AF647)-conjugated mouse anti-human CD9 (1:10; Exbio).

Techniques: Multiplex Assay, Fluorescence, Purification, Labeling, Staining, Double Staining